Prevalence and molecular detection of blood protozoa in domestic pigeon

The present study was carried out to know the status of haemoprotozoan infection of domestic pigeon in Assam by microscopic examination of blood of pigeons for a period of one year which revealed an overall prevalence of 53.39%. Three species viz. Haemoproteus columbae (29.93%), Plasmodium relictum (21.29%) and Leucocytozoon sp. (2.16%) were identified either in single or mixed infection. According to age, highest prevalence was recorded in adult (61.81%) and lowest in squab (36.25%). Comparatively, infection was recorded higher in females (58.22%) than males (48.79%). Season wise, infection was recorded highest during Pre-monsoon (72.22%) and lowest during Postmonsoon. Amplification of cyt b gene of Haemoproteus columbae in positive samples by PCR showed clear band at 207 bp. Amplification of mt- cyt b gene of Haemoproteus spp. and Plasmodium spp. by PCR on positive samples revealed clear band at 525 bp.

Original Research Article https://doi.org/10.20546/ijcmas.2019.805.163

Prevalence and Molecular Detection of Blood Protozoa in Domestic Pigeon

Munmi Saikia 1 *, Kanta Bhattacharjee 1 , Prabhat Chandra Sarmah 1 , Dilip Kr Deka 1 ,

Shantanu Tamuly 2 , Parikshit Kakati 1 and Pranab Konch 3

1

Department of Parasitology, 2 Department of Biochemistry, 3 Department of Pathology, College of Veterinary Science, Khanapara, Guwahati-781022, Assam, India

*Corresponding author

A B S T R A C T

Introduction

Species of apicomplexan Haemoproteus,

Plasmodium and Leucocytozoon are well

known genera of avian haematozoa and

comprise a diverse group of vector

transmitted parasites

They are closely related genetically but

different in life history traits (Valkiunas,

1993) Avian malaria, caused by Plasmodium

sp is transmitted to birds by mosquitoes and

has a long-term effect on the reproductive

system of the host causing population

decrease (Lapointe et al., 2012)

Leucocytozoon sp typically causes anaemia and enlargement of liver and spleen (Dey et al., 2010) Haemoproteus columbae

commonly infect pigeon and doves and is widely distributed in tropical and subtropical regions and transmitted by blood sucking

hippoboscid fly Pseudolynchia canariensis Its pathogenicity is generally low; however,

due to acute infections in severely affected

young pigeon heavy mortality is seen (Dey et al., 2010)

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 8 Number 05 (2019)

Journal homepage: http://www.ijcmas.com

The present study was carried out to know the status of haemoprotozoan infection of domestic pigeon in Assam by microscopic examination of blood of pigeons for a period of one year which revealed an overall prevalence of 53.39% Three species viz

Haemoproteus columbae (29.93%), Plasmodium relictum (21.29%) and Leucocytozoon sp

(2.16%) were identified either in single or mixed infection According to age, highest prevalence was recorded in adult (61.81%) and lowest in squab (36.25%) Comparatively, infection was recorded higher in females (58.22%) than males (48.79%) Season wise, infection was recorded highest during Pre-monsoon (72.22%) and lowest during

Post-monsoon Amplification of cyt b gene of Haemoproteus columbae in positive samples by PCR showed clear band at 207 bp Amplification of mt- cyt b gene of Haemoproteus spp and Plasmodium spp by PCR on positive samples revealed clear band at 525 bp

K e y w o r d s

Pigeon,

Haemoprotozoa,

Prevalence, PCR,

Assam

Accepted:

12 April 2019

Available Online:

10 May 2019

Article Info

Materials and Methods

Study period

The present study was undertaken to ascertain

the haemoprotozoan infection in domestic

pigeon (Columba livia domestica) for a period

of one calendar year w.e.f February 2015 to

January 2016

Sample collection

Four districts of Assam namely Kamrup

Rural, Kamrup Metro, Lakhimpur and

Dhemaji formed the study areas Blood

samples of pigeons were collected from

different households, market places and

temple premises

The pigeons were categorized according to

age viz squab (< 30 days), young (30-90

days) and adult (> 90 days) and sex (male and

female) The study period was divided into

four seasons viz Pre-monsoon (March, April,

May), Monsoon (June, July, August,

September), Post-monsoon (October,

November) and Winter (December, January,

February)

Sampling of Blood for Detection of

Haemoprotozoa

Blood samples from 324 live pigeons were

collected from wing vein using a 2 ml

disposable syringe in EDTA vials and brought

to the laboratory for parasitological and

molecular analysis For molecular analysis,

the anticoagulated blood was stored in deep

freeze at –20 ºC until further use thin blood

smears were prepared using commercial

Giemsa stain and examined under high power

(40X) and oil immersion objective (100X) of

light microscope for detection of

Haemoproteus sp and Plasmodium sp inside

the red blood cells and Leucocytozoon sp

inside the lymphocytes and monocytes The

parasites were identified on the basis of their characteristic morphology (Levine, 1977; Soulsby, 1982) and percent parasitaemia (No

of parasitized cell /Total no of respective cell

x 100 = % parasitaemia) in positive cases were estimated

Molecular detection of Haemoproteus columbae

DNA was extracted from 30 random positive samples of blood using DNeasy Blood and Tissue Kit (Qiagen Germany) as per manufacture’s guidelines The extracted DNA was stored at -20º C until further use The PCR was performed following the method of

Doosti et al., (2014) to amplify a segment of cyt b gene of Haemoproteus columbae using oligonucleotide primer (H clom- F 5′-TTA

GAT ACA TGC ATG CAA CTG GTG-3′and

H clom-R 5′-TAG TAA TAA CAG TTG

CAC CCC AG-3′) in 25µl reaction mixture containing 1µl DNA template, 1µl (20 pmol/ µl) of each forward and reverse primer, 1µl MgCl2 (50 mM), 0.5µl dNTPs mix (10 mM), 0.25µl Taq DNA polymerase (5 IU/ µl) and the remaining volume adjusted with nuclease free water PCR amplification was performed

in a Technee-5000 thermal cycler (Bibby Scientific) PCR was performed with Initial denaturation at 94˚C for 5 min followed by 30 cycles consisting of denaturation at 94˚C for 1 min, annealing at 60º C for 1 min, extension

at 72˚C for 1 min and final extension at 72˚C for 5 min A negative control consisting of a reaction mixture without the DNA was used

Molecular detection of Haemoproteus spp and Plasmodium spp

DNA was extracted from 10 random positive blood samples of pigeons having

simultaneous infection of Haemoproteus columbae and Plasmodium relictum on blood

smear examination using DNeasy Blood and Tissue Kit (Qiagen Germany) PCR was

performed following the method described by

Valkiunas et al., (2008) with little

modification to amplify a segment of

mitochondrial cyt b gene of Haemoproteus

spp and Plasmodium spp using

oligonucleotide primers (Haem F

5′-ATGGTGCTTTCGATATATGCATG-3′ and

5′-GCATTATCTGGATGTGATAATGGT-3′)

PCR amplification was done in a

Technee-5000 thermal cycler (Bibby Scientific) in 25µl

reaction mixture containing 5 µl of genomic

DNA, 2.5 µl 10x PCR buffer, 1.0 µl MgCl2

(50 mM), 0.5 µl dNTP (10 mM), 1.0 µl (20

pmol/ µl) of each forward and reverse primer,

0.2 µl Taq DNA polymerase (5 IU/ µl) and

nuclease free water up to 25 µl PCR

amplification was done with initial

denaturation at 94˚C for 3 min, and then 35

cycles consisting of denaturation for 30 sec at

94˚C, annealing for 30 sec at 50˚C and

extension for 45sec at 72˚C, followed by final

extension at 72˚C for 10 min A negative

control consisting of a reaction mixture

without the DNA template was taken

Electrophoresis

For visualization of the PCR product, gel

electrophoresis of amplified DNA was done

in 1.5 % agarose gel for 1 hour at 5 Volts per

cm using 1 X Tris Acetate EDTA (1X TAE)

running buffer Four µl of the PCR product

mixed with 3 µl of gel loading dye (6X DNA

Loading Dye, Fermentas) was loaded on to

the gel with standard markers (100 bp DNA

ladder, Fermentas) The gel was then stained

with ethidium bromide (0.5 µg/ ml) and

visualized under gel doc (DNR Bio-Imaging

System, Mini Lumi) for the expected product

size and images were obtained

Statistical analysis

Chi-square test was used for statistical

analysis of the prevalence data using SAS

v.20 software

Results and Discussion

Prevalence of haemoprotozoa according to parasite species

Species wise, prevalence of Haemoproteus columbae was 29.93%, Plasmodium relictum 21.29% and Leucocytozoon sp 2.16% (Table

1 and Fig 1) without significant statistical difference (P<0.05)

Prevalence of H columbae ranging from 15

to 80% was reported by several workers (18%

by Ishtiaq et al., 2007 from India; 22% by Valkiunas et al., 2008; 47.05% by Radfar et al., 2011 from Iran; 60% by Roy et al., 2011 from Assam; 69.09% by Varshney et al.,

2014 from Surat; 74.28% Borkataki et al.,

2015 from Jammu) Report of 28%

prevalence by Abed et al., (2014) from Iraq is

in agreement with our findings Studies to date have reported that the most common

blood parasite found in pigeons is H columbae, usually considered to be non

-pathogenic but may cause disease in stressed pigeons The variation in prevalence rate of this parasite in different countries might be influenced by geographical region, vector abundance, host genotype, host size, age or sex of host, feeding habitats, health status of bird etc

Prevalence of Plasmodium relictum (27.5%)

similar to our findings was reported from

Kamakhya premises, Assam by Roy et al., (2011) and Gupta et al., (2011) from Uttar

Pradesh (6.76%) This finding substantiates

that mosquito of Genus Culex, the vector of

pigeon malaria is commonly prevalent in Assam

Prevalence of Leucocytozoon sp in the

present finding is similar to the report of 2%

by Nath et al., (2014) from Bangladesh

However, higher prevalence has been reported by several workers (6.4% by Natala

et al., 2009; 20% by Dey et al., 2010 and 25%

by Valkiunas et al., 2008) which contradict

our findings and possibly it might be due to

study made in different environments,

population of vector fly and number of birds

examined In the present study, prevalence of

mixed infection of Haemoproteus columbae

and Plasmodium relictum was recorded as

7.71% Beadell et al., (2009) similarly

reported 6.8% pigeons in the Australo-Papuan

region Contrary to our finding, slightly lower

prevalence (2.67%) was reported by Jahan et

al., (2011) from Uttar Pradesh Co-infection

with two or more parasites revealed that the

presence of one haemoparasite predisposes to

other haemosporidian infections Our finding

agrees with the above statement There was a

noticeable relationship between the

prevalence of H columbae (29.93%) and its

vector, Pseudolynchia canariensis (15.12%)

The closeness in their percentage prevalence

suggests that most of the vector harboured by

the pigeons were probably carrying

pathogens According the Taylor et al.,

(2007), the presence of Plasmodium and

Leucocytozoon in the blood of the pigeons

was an indication of the presence of Culex

and Simulium respectively, as they are

established vectors of these haemoparasites

In our study of haemoprotozoa, the mean

concentration of parasites was 1-6 pars/100

RBC for both H columbae and P relictum

with variation in the shape and size of the

gametocytes (Fig 5) Similar reports were

made (Gupta et al., 2011; Jahan et al., 2011

and Hussein et al., 2016) from India and

elsewhere

Age wise prevalence of haemoprotozoan

parasites

The present finding recorded 61.81%

prevalence of haemoprotozoa in adult

followed by young (56.71%) and squab

(36.25%) (Table 2 and Fig 2) with statistical

significance (P<0.05) Our report conform

that of Momin et al., (2014) from Bangladesh

who stated that adults were 6.89 times more

susceptible than young birds Msoffe et al.,

(2010) from Tanzania also made identical report It is apprehended that adult birds are generally more attacked by vector flies

Sex wise prevalence of haemoprotozoa parasites

Sex wise, prevalence was recorded more in female (58.22%) than the male (48.71%) (Table 3, Fig 3) with non-significant (P>0.05) difference and agreeing with the

findings of Momin et al., (2014) However, several workers from abroad (Dey et al., 2010; Opara et al., 2012 and Hussein et al.,

2016) recorded higher prevalence in male than female Though the exact cause of higher infection in females could not be explained it was assumed due to higher level of prolactin and progesterone suppressing the immune system of the individual and making the female more susceptible to any infection

Seasonal prevalence of haemoprotozoa

Haemoprotozoan infection was recorded highest during Pre-monsoon season (72.22%) and lowest during Post monsoon, however, infection was more or less present throughout the year (Table 4 and Fig 4) It might be due abundance of vector in Pre monsoon season Literature is scant leading to less information

on this aspect

Molecular detection of Haemoproteus columbae

PCR employed for molecular detection of H columbae by amplification of cyt b gene

showed clear band at 207 bp (Fig 6a) similar

to the work of Doosti et al., (2014) who reported 23.18% prevalence of H columbae

in 220 pigeons in Iran

Table.1 Species-wise prevalence of haemoprotozoa in pigeon

Chi-square value

Single infection

No (%)

Mixed infection

No (%)

Total

No (%)

H columbae P relictum Leucocytozoon sp

Haemoproteus

columbae

69 (21.29)

(7.71)

3 (0.92)

97 (29.93)

23.6716*

Plasmodium

relictum

44 (13.58)

25 (7.71)

(0.0)

69 (21.29)

Leucocytozoon

sp

4 (1.23)

3 (0.92)

0 (0.0)

(2.16)

Overall 117

(36.11)

28 (8.64)

25 (7.71)

3 (0.92)

173 (53.39)

*P(<0.05)

Table.2 Age wise prevalence of haemoprotozoan parasites in pigeon

Age group

(No

examined)

Parasite Prevalence Total

No (%)

Chi-square value

Haemoproteus columbae

No positive (%)

Plasmodium relictum

No Positive (%)

Leucocytozoon

sp

No Positive(%)

Squab (80)

(< 30 days)

15 (18.75)

14 (17.50)

0 (0.0)

29 (36.25)

24.6516*

Young (134)

(30-90 days)

46 (34.32)

26 (19.40)

4 (2.98)

76 (56.71) Adult (110)

(>90 days)

36 (32.72)

29 (26.36)

3 (2.72)

68 (61.81) Total (324) 97

(29.93)

69 (21.29)

7 (2.16)

173 (53.39)

* P(<0.05)

Table.3 Sex-wise prevalence of haemoprotozoan parasites in pigeon

Sex No

examined

Haemoproteus columbae

Plasmodium relictum

Leucocytozoon

sp

Total

Chi-square value

No Positive (%) No positive (%) No positive (%) No positive (%)

(25.30)

36 (21.68)

3 (1.80)

81 (48.79)

1.3215NS

(34.81)

33 (20.88)

4 (2.53)

92 (58.22)

(29.93)

69 (21.29)

7 (2.16)

173 (53.39)

NS (Non significant), P>0.05

Table.4 seasonal prevalence of haemoprotozoa in pigeon

Month/season Samples screened

for haemoprotozoa

Sample positive for haemoprotozoa (%)

Chi-square value Premonsoon (March,

April, May)

(72.22%)

6.7118NS

Monsoon (June, July,

August, September)

(46.15)

Post monsoon

(October, November)

(53.57)

Winter (December,

January, February)

(42.0)

(53.39)

NS (Non-significant) P(>0.05)

Fig.1 Species-wise prevalence of haemoprotozoa in pigeons

Leucocytozoon sp

Fig.2 Age-wise prevalence of haemoprotozoa in pigeons

Fig.3 Sex-wise prevalence of haemoprotozoan parasites in pigeon

Fig.4 Seasonal prevalence of haemoprotozoa in pigeon

Fig.5 Immature stages (gametocytes) (a-b), mature gametocytes (c-f), of Haemoproteus

columbae; mature gametocytes (g-h), of Plasmodium relictum 1000X (Oil immersion)

b

a

d

c

f

e

h

g

Fig.6 (a) PCR product at 207 bp of Haemoproteus columbae ( L-Ladder 100 bp, Lane-1, 2, 3, 4,

5, 6 & 8- positive sample, 7- Negative control) & b) PCR product at 525 bp of Haemoproteus and Plasmodium ( L-Ladder:100 bp, Lane-1, 2 , 3, 4 & 5 -positive samples and 6-Negative

control)

Molecular detection of Haemoproteus spp

and Plasmodium spp

PCR employed for simultaneous detection of

H columbae and P relictum by amplification

of mt-cyt b gene revealed clear band at 525 bp

(Fig.6b) In our present study, by microscopic

examination some early developmental stages

of Haemoproteus columbae and Plasmodium relictum could not be morphologically

differentiated, especially in mixed infection However, it was confirmed by PCR

Similarly, Hellgren et al., (2004) opined that

by conventional microscopy, especially in

chronic infections, species of Haemoproteus

might be difficult to distinguish from avian

207bp

L 1 2 3 4 5 6 7 8

1 2 3 4 5 6

L

525 bp

species of Plasmodium Several PCR-based

methods for studies of Haemoproteus spp

and Plasmodium spp have been reported

(Bensch et al., 2000; Richard et al., 2002;

Bell et al., 2015) Similarly, Hellgren et al.,

(2004) and Bell et al., (2015) described a

Nested PCR assay targeting the cyt b gene of

the parasites, for screening and typing of

Leucocytozoon sp in parallel with

Haemoproteus and Plasmodium in avian

blood samples From the present study, it was

found that 53.39% pigeon were infected with

three types of blood protozoa such as

Haemoproteus columbae (29.93%),

Plasmodium relictum (21.29%) and

Leucocytozoon sp (2.16%) It may be

concluded that the protozoan infections in

pigeon are highly endemic in Assam The

systematic study conducted for the first time

in Assam led to a significant conclusion that

favourable climatic condition and presence of

vectors are the contributing factors towards

prevalence of haemoprotozoan parasites

Acknowledgement

The authors are thankful to the Dean, College

of Veterinary Science, Assam Agricultural

University for providing the necessary

facilities to conduct the study

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